The invention relates to a novel recombinant DNA coding for a novel protein with endochitinase activity or for a precursor thereof, to a bacterium, a yeast or a fungus containing this recombinant DNA, to a plant cell, a plant or a part of a plant, in particular a plant seed, containing this recombinant DNA, to a method of rendering plants resistant to pathogens such as fungi and bacteria, as well as arthrepods, especially insects, and nematodes, which comprises a step involving transformation by this gene, to this novel protein and to a process for its preparation.
Cultivated plants are subject to attacks by pathogens such as fungi and bacteria, as well as arthropods, especially insects, and nematodes, which are responsible for substantial losses of harvest. At the present time, the main means of controlling these pathogens is to use chemical substances with fungicidal or bactericidal activity. It is now known that plants react naturally to these attacks by means of various defense mechanisms which unfortunately are generally triggered too late and are too weak in intensity to be sufficiently effective. One of these mechanisms includes the induction of an enzyme called chitinase EC 3.2.1.14 (A. Toppan et al., 1982, Agronomie, 2, 829-834). This induction can be stimulated artificially by substances such as ethylene, the consequence of which is to increase the resistance of the treated plant to pathogens (Boller T., 1988, Oxford Surveys of Plant Molecular and Cell Biology, 5, 145-174).
Chitin is a linear polysaccharide polymer consisting of N-acetylglucosamine units joined by .beta.-1,4 linkages. Said polymer is a structural compound present in the wall of most pathogenic fungi, in the exoskeleton of arthropods, especially insects, and in the outer envelope of the eggs and cysts of nematodes. Enzymes called chitinases are capable of degrading chitin. They are divided up into two different groups defined according to their mode of attacking chitin: exochitinases, which are capable of freeing the N- acetylglucosamine unit located at the non-reducing ends of the chains, and endochitinases, which are capable of fragmenting the chains and are the only chitinases capable of inhibiting the growth of mycelial hyphae in vitro (Roberts W. K. et al., 1988, Gen. Microbiol., 134, 169-176). The vast majority of the known plant chitinases are of the endo type, in contrast to the known bacterial chitinases, which are of the exo type (Roberts W. K. et al., 1988, Gen. Microbiol., 134, 169-176).
DNA sequences coding for bacterial exochitinases have already been isolated and cloned (Jones J. D. G. et al., 1986, EMBO J., 5, 467-473, and Sundheim L. et al., 1988, Physiol. Molec. Plant Pathol., 33, 483-491). U.S. Pat. No. 4,751,081 describes the isolation and cloning of the complete gene coding for the chitinase of Serratia marcescens and the transformation of the bacteria Pseudomonas fluorescens NZ130 and Pseudomonas putida MK280 by this gene. These transformed bacteria are capable of slightly degrading a colloidal chitin dispersed in the bacterial culture medium. The work of Harpster M. H. et al., 1989, Nucl. Ac. Res., 17, 5395, has shown that this gene codes for an exochitinase, which explains the low degradation efficacy observed (cf. Table 2, columns 13 and 14 of said document). The publication by Jones J. D. G. et al. (1988), Mol. Gen. Genet., 212, 536-542, mentions the transformation of tobacco plants by Agrobacterium tumefaciens containing a chimeric gene comprising the coding part of the exochitinase of Serratia marcescens, under the control of different plant promoters. Said document gives no indication of any increase in the pathogen resistance conferred by the expression of this exochitinase.
Genomic DNA and/or complementary DNA sequences coding for certain plant endochitinases have also been isolated and cloned (Bfoglie K. E., 1986, Proc. Ntl. Acad. Sci. USA, 83, 6820-6824, and Hedrick S. A., 1988, Plant Physiol., 86, 182-186).
International patent application 90/07001 describes the construction of a plasmid carrying a complementary DNA of the endochitinase of the bean Phaseolus vulgaris under the control of a strong promoter, transformation with the aid of Agrobacterium tumefaciens, the regeneration of the transformed tobacco, experiments showing the enhanced resistance of the regenerated plants to the fungi Rhizoctonia solani and Botrytis cinerea, the production of transgenic tomato plants expressing the bean chitinase and the production, with the aid of this gene, of transgenic colza plants having an enhanced level of chitinase activity and an enhanced resistance to Rhizoctonia solani compared with the non-transformed colza plants.
A genomic DNA and/or complementary DNA sequence coding for a chitinase of a filamentous fungus has never been isolated hitherto. The only partially isolated and characterized chitinase of a filamentous fungus is the endochitinase of Aphanocladium album (Kuntz, 1991-- Doctoral Thesis at the Universite de P. et M. Curie--Paris).